Natural drugs for the treatment of inflammation and melanoma

ABSTRACT

The invention is directed to natural drug compositions comprising turmeric, boswellia and ginger. Methods for the use of the inventive drug compositions in the treatment of inflammatory disorders and melanoma are within the scope of the invention.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of application Ser. No. 15/829,716filed Dec. 1, 2017, which claims priority under 35 U.S.C. § 119(e) toProvisional Application No. 62/435,608, filed Dec. 16, 2016. The entirecontents of these applications are incorporated herein by reference forall purposes.

FIELD OF THE INVENTION

The invention is in the field of drug compositions derived from naturalsources.

BACKGROUND

Inflammation can be considered a central feature of manypathophysiological conditions that are initiated in response to tissueand cellular damage by pathogens, noxious stimuli, such as chemicals andphysical injury. Such damage leads to the secretion of cytokines andother mediators as well as activation and migration of immune cells.These mediators add to the generation of excess free radicals such asreactive oxygen species (ROS) and reactive nitrogen species (RNS) whichlead to DNA damage. Acute inflammation is a short-term response thatusually results in healing as leukocytes infiltrate the damaged region,removing the stimulus and repairing the tissue. Chronic inflammation, bycontrast, is a prolonged, dysregulated and maladaptive response thatinvolves active inflammation and tissue destruction. Such persistentinflammation is associated with many chronic human conditions anddiseases, including allergy, atherosclerosis, cancer, arthritis andautoimmune diseases. Inflammation is currently treated by NSAIDs(non-steroidal anti-inflammatory drugs). Unfortunately, these drugs leadto blood clots and consequently increase the risk for heart attacks andstrokes. Natural products are rich source for discovery of new drugsbecause of their chemical diversity. Natural products from medicinalplants may play a major role in treating many diseases associated withinflammation.

SUMMARY OF THE INVENTION

The invention provides natural drug compositions developed fromturmeric, boswellia and ginger. The invention further provides methodsfor using such drug compositions in the treatment of inflammatorydisorders and melanoma.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the cytotoxic effect of an embodiment of the drugcomposition on RAW 264.7 cells using an MTT assay.

FIG. 2 shows the inhibitory effect of an embodiment of the drugcomposition on NO production in LPS-stimulated RAW 264.7 cells. Theproduction of NO was assayed in the culture medium of cells stimulatedwith LPS (1 μg/ml) for 24 h in the presence of the drug composition atdifferent concentrations.

FIG. 3 shows the cytotoxic effect of an embodiment of the drugcomposition on melanoma cells using an MTT assay.

FIG. 4. Shows the morphological changes observed in B16-F10 melanomacells treated with an embodiment of the drug composition. Cells weretreated with 0.4 and 1 mg/mL of the drug composition for 48 hr.

DEFINITIONS

As used herein, the term “active agent” refers to the components of theinventive drug composition which have a desired biological orpharmacological effect in a subject. The term “active agent” includesturmeric, boswellia, ginger, or combination thereof.

As used herein, the term “about” or “approximately” refers to aquantity, level, value, number, frequency, percentage, dimension, size,amount, weight or length that is the same as the stated value, or thatvaries by as much as 30, 25, 20, 25, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1% tothe referenced quantity, level, value, number, frequency, percentage,dimension, size, amount, weight or length. In particular embodiments,the terms “about” or “approximately” when preceding a numerical valueindicates the stated value plus or minus a range of 15%, 10%, 5%, or 1%,or any intervening range thereof.

As used herein, the term “bioavailability” refers to the degree and rateat which a substance is absorbed into a living system or is madeavailable at the site of physiological activity.

As used herein, the term “bioenhancer” refers to a substance thatincreases the bioavailability of a material that has a biological orpharmacological effect in a subject compared to the bioavailability ofthe material in the absence of the substance.

As used herein, the term “boswellia” refers to any material obtainedfrom a genus of trees and shrubs in the order Sapindales, known fortheir fragrant resin. Boswellia includes materials obtained from B.sacra (synonyms B. carteri and B. bhaw-dajiana), B. frereana, B.papyrifera, and B. serrata, B. ameero, B. bullata, B. dalzielii, B.dioscorides, B. elongata, B. nana, B. neglecta, B. ogadensis, B.pirottae, B. popoviana, B. rivae, B. socotrana, and mixtures thereof.Such materials can be obtained from the leaves, flowers, stems, roots,resin, and/or bark of such trees and shrubs. Boswellia includes anextract, fresh and/or dried material obtained from the leaves, flowers,stems, roots, resin, and/or bark of such trees and shrubs. Boswelliaincludes acids derived from the resin of such trees and shrubs.

As used herein, the term “effective concentration” or “effective amount”is used to describe an amount or concentration of an active agent ordrug composition according to the present invention which is used toproduce an intended result. In the case of the present invention,effective concentrations are generally concentrations which areeffective to treat an inflammatory disorder or melanoma.

As used herein, the term “ginger” refers to any material obtained fromthe flowering plant Zingiber officinale. Ginger for use with theinvention includes, but is not limited to, raw ginger, ginger oil,ginger extract, gingerols, or a combination thereof. Ginger includes,but is not limited to, ginger, ginger oil, ginger extract, gingerols, ora combination thereof, derived from the root of the ginger plant.

As used herein, the term “lecithin,” refers to any or all of thephosphatides, pure or in blends comprising phosphatidylcholine,phosphatidylethanolamine, phosphatidylinositol, and/orphosphatidylserine, and/or other phosphatides regarded as lecithins.

As used herein, the terms “treating” or “treatment” or “to treat” or“alleviating” or “to alleviate” refer to both 1) therapeutic measuresthat cure, slow down, lessen symptoms of, and/or halt progression of apathologic condition or disorder and 2) prophylactic or preventativemeasures that prevent and/or slow the development of a targetedpathologic condition or disorder. Thus, those in need of treatmentinclude those having the disorder; those prone to have the disorder; andthose in whom the disorder is to be prevented.

As used herein, the term “turmeric” refers to any material obtained fromthe herb Curcuma longa. Turmeric for use with the invention includes,but is not limited to, raw turmeric, turmeric oil, turmeric extract, ora combination thereof.

DETAILED DESCRIPTION

The invention generally relates to drug compositions developed fromnatural sources. More particularly, the invention relates to drugcompositions developed from synergistic combinations of turmeric,boswellia and ginger. Methods of using the drug compositions for thetreatment of inflammatory disorders and melanoma are also within thescope of this invention.

In one aspect, the invention provides a drug composition comprising atleast one of turmeric, boswellia and ginger. One or more of theturmeric, boswellia and ginger can be in the form of fresh or driedmaterial. Dried material can be in the form of a powder, for example.The drug compositions can comprise extracts of at least one of turmeric,boswellia and ginger. The drug composition can comprise: turmericextract and boswellia extract; turmeric extract and ginger extract;boswellia extract and ginger extract; or turmeric extract, boswelliaextract, and ginger extract.

The relative amounts of each extract may vary depending on the disorderthat is targeted for use of the drug composition. The drug compositioncan comprise, for example, about 50% turmeric, about 30% boswellia, andabout 20% ginger. It is further contemplated that these percentages, mayvary (+/−) up to 80%. Thus, the drug composition can comprise about 95%to about 30% turmeric, about 54% to about 6% boswellia, and about 36% toabout 20% ginger. It is further understood that the percentagesdisclosed in this paragraph may vary, or be offset, by an amountcorresponding to the amount of any bioenhancer that is added to increasethe bioavailability of the turmeric, boswellia, and/or ginger (i.e.active agents).

The drug compositions of the invention can comprise one or more ofturmeric extract, boswellia extract, and ginger extract. Turmericextract may be standardized to contain about 95-98% curcuminoids.Turmeric extracts for use with the invention may be standardized tocontain curcuminoids in an amount of about 100%, 99% 98%, 97%, 96%, 95%,94%, 93%, 92%, 91%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, or anyamount intervening these amounts. Ginger extract may contain gingerols,including 8-gingerol, 10-gingerol, and 12-gingerol. Ginger extracts foruse with the invention may be standardized to contain one or more ofthese gingerols in an amount of about 100%, 99% 98%, 97%, 96%, 95%, 94%,93%, 92%, 91%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, 5%, 2%, orany amount intervening these amounts. In a non-limiting embodiment, theginger extract is standardized to about 5% of one or more gingerols. Inanother non-limiting embodiment, the ginger extract is standardized toabout 20% of one or more gingerols.

Boswellia for use with the drug composition can comprise boswelliaextract. The boswellia extract can be standardized for boswellic acid.Boswellia extracts can be standardized to contain one or more boswellicacids in amount of about 100%, 99% 98%, 97%, 96%, 95%, 94%, 93%, 92%,91%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, 5%, or any amountintervening these amounts. In one non-limiting embodiment, the boswelliaextract is standardized to about 30% boswellic acid. The boswellic acidto which the boswellic extract is standardized can beacetyl-11-keto-beta-boswellic acid (AKBA).

Boswellia for use with the invention can be boswellia gum resin. The gumresin of boswellia is a complex mixture comprising: boswellia oilfraction (BOIL) containing essential oil/boswellia volatile oil fraction(BVOIL); non-acidic boswellia low polar gum resin extract fraction(BLPRE); boswellic acids (BA); and sugars and polysaccharide fraction(BSUG). The boswellia gum resin can be standardized to contain one ormore of BOIL, BVOIL, BLPRE, BA and BSUG in an amount of about 100%, 99%98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 80%, 70%, 60%, 50%, 40%,30%, 20%, 10%, or any amount intervening these amounts. It iscontemplated that the drug composition can be formulated with boswelliain the form of BIOL, BVOIL, BLPRE, BA, BSUG, or a combination thereof.It is contemplated that the drug composition can be formulated withboswellia in the form of purified BIOL, purified BVOIL, purified BLPRE,purified BA, purified BSUG, or a combination thereof.

In a specific, non-limiting example, the drug composition of theinvention comprises about 50% turmeric extract standardized to about 95%curcuminoids, about 30% boswellia extract standardized to about 30%AKBA, and about 20% ginger extract standardized to about 20% gingerols.In another embodiment, the drug composition comprises: 100 mg to 10,000mg of turmeric standardized to about 25% to about 99% curcuminoids; 100mg to 10,000 mg boswellia standardized to about 5% to about 99% AKBA;and 100 mg to 10,000 mg of ginger standardized to about 5% to about 99%gingerols. In another embodiment, the drug composition can comprise 250mg turmeric extract standardized to about 95% curcuminoids, 150 mg.boswellia standardized to about 30% AKBA, and 100 mg ginger standardizedto about 20% gingerols.

In some aspects, the drug compositions are formulated for administrationto a subject, such as a human subject. Such formulations may assume anyform suitable for oral administration to a subject including, but notlimited to pills, capsules, tablets, liquids (e.g. beverages, or drops),gummies, pastes, emulsions, drops, syrups, gels, softgels, powders, orfood supplements. The invention also contemplates formulating the drugcompositions as cosmeceuticals, including, but not limited to, creams,gels, powders, milks, emulsions, liquids, sprays, foams, sticks andpastes. The drug compositions can be formulated for administration byany suitable means, including topically, orally, sublingually, buccally,intra-ocularly, intravenously, intramuscularly, intra-arterially, bysuppository, intranasally, subcutaneously, parenterally, intravaginally,rectally, or by inhalation. Thus, the drug compositions may beformulated with a suitable pharmaceutical carrier (e.g. artificialpharmaceutical carriers) for administering according to any of thepreceding routes of administration. The drug compositions can compriseone or more carriers including, but not limited to, sodium citrate,dicalcium phosphate, fillers or extenders (such as starches, lactose,sucrose, glucose, mannitol, and silicic acid), binders (such as, forexample, carboxymethyl-cellulose, alginates, gelatin,polyvinylpyrrolidinone, sucrose, and acacia), disintegrating agents(such as agar-agar, calcium carbonate, potato or tapioca starch, alginicacid, silicates, and sodium carbonate), buffering agents andcombinations thereof.

The drug compositions can be in a dosage form that includes but is notlimited to powders, pills, tablets, pellets, capsules, thin films,solutions, sprays, syrups, linctuses, lozenges, pastilles, chewing gums,pastes, vapours, suspensions, solutions, emulsions, ointments, creams,lotions, liniments, gels, drops, topical patches, buccal patches, beads,gummies, gels, sols, injections and the like. The drug composition cancomprise at least one pharmaceutically acceptable excipient. Suitableexcipients for use with the drug compositions include, but are notlimited to, those disclosed in Remington's Pharmaceutical Sciences, 18thed. the disclosure of which is incorporated herein by reference in itsentirety for all purposes. The pharmaceutically acceptable excipient canbe an artificial pharmaceutical carrier.

In some aspects of the invention, the drug composition is formulated toachieve increased bioavailability of one or more of the active agents inthe drug composition. As used herein, the phrase “increasingbioavailability” refers to increasing the bioavailability of one or moreactive agents in the drug composition relative to a control set ofconditions. The term “active agent” as used herein includes turmeric,boswellia and ginger. Active agents in the drug composition furtherinclude the constituent compounds included in the turmeric, boswelliaand/or ginger. Constituent compounds in turmeric include, but are notlimited to, curcuminoids. As used herein, the term “curcuminoids” refersto curcumin, demethoxycurcumin, and bisdemethoxycurcumin. Constituentcompounds in ginger include, but are not limited to gingerols, including8-gingerol, 10-gingerol, and 12-gingerol. Constituent compounds inboswellia include, but are not limited to, BIOL, BVOIL, BLPRE, BA, BSUG,AKBA, or a combination thereof.

The drug composition can be formulated with a bioenhancer to achieveincreased bioavailability of the active agents. Accordingly, the drugcomposition can comprise at least one of turmeric, boswellia, ginger,and an amount of bioenhancer effective to increase the bioavailabilityof at least one of the turmeric, boswellia and ginger. The drugcomposition can comprise turmeric extract, boswellia extract, gingerextract, and an amount of bioenhancer sufficient to increase thebioavailability of at least one of the turmeric extract, boswelliaextract, and ginger extract. In some aspects, the bioenhancer ispurified. As used herein, the term “purified” refers to a compoundhaving been separated from a component of the composition in which itwas originally present. The bioenhancer can be present in the drugcomposition in an amount of about 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%,1%, 0.5%, or 0.25%, with the remaining portion of the composition beingturmeric, boswellia and ginger. The bioenhancer can be present in thedrug composition in an amount of about 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%,2%, 1%, 0.5%, or 0.25%, with the remaining portion of the compositionbeing turmeric extract, boswellia extract and ginger extract. The drugcomposition can comprise 1% bioenhancer, with the remaining portion ofthe composition comprising turmeric, boswellia and ginger. In anon-limiting embodiment, the drug composition comprises 1% bioenhancer,with the remaining portion of the composition comprising turmericextract, boswellia extract and ginger extract. It will be understoodthat the amount of the one or more of the active agents (e.g. turmeric,boswellia and ginger) as disclosed herein will be reduced by thecorresponding amount of amount of bioenhancer that is used forincreasing the bioavailability of the active agents. For example, a drugcomposition comprising 50% turmeric, 30% boswellia, and 20% ginger maycomprise 49% turmeric, 30% boswellia, 20% ginger, and 1% bioenhancer.

Bioenhancers for use with the invention include, but are not limited to,lecithin, turmeric oil, boswellia oil, ginger oil (e.g. ginger rootoil), or a combination thereof. The turmeric oil, boswellia oil andginger oil can be volatile oils, non-volatile oils, or a combinationthereof.

Lecithins for use with the drug compositions can be edible lecithins.Edible lecithins are well-known, widely available, and are described anddefined in detail in the public literature. For example, they aredescribed in: Kirk Othmer, Encyclopedia of Chemical Technology, Volume14, pp. 250-269; in the Encyclopedia of Food Science, Peterson andJohnson, editors, Avi Publishing Co. 1978, pp. 461, 467; and LECITHINS,edited by Bernard F. Szuhaj, and Gary R. List, which was published bythe American Oil Chemists' Society as a monograph. (Also see especiallyChapter 8, Commercial Lecithin Products; Food Use of Soybean Lecithin,by W.E. Prosise). The descriptions of all these references areincorporated by reference herein in their entirety and for all purposes.Lecithin for use with the drug compositions may be sunflower lecithin.

In a non-limiting embodiment wherein the drug composition is formulatedwith bioenhancers, the drug composition can comprise (i) a turmericcomponent comprising about 98% turmeric extract (standardized to about95% curcuminoids), about 1% lecithin, and about 1% turmeric oil, (ii) aboswellia component comprising about 98% boswellia extract (standardizedto about 30% of one or more boswelic acids), about 1% boswellia oil, andabout 1% lecithin, and (iii) a ginger component comprising about 98%ginger extract (standardized to about 20% gingerols), about 1% gingeroil, and about 1% lecithin. In such embodiments, the drug compositioncan comprise about 50% of the turmeric component, about 30% of theboswellia component, and about 20% of the ginger component. It will beunderstood that in embodiments where one or more of the extractscontains oil of the extracted base material, the amount of oil used as abioenhancer will be in addition to the oil contained in the extract. Forexample, wherein the drug composition comprises boswellia extractcontaining boswellia oil, the drug composition can contain 1% boswelliaoil in addition to the boswellia oil contained in the extract. It willbe understood that the lecithin, boswellic acid(s), boswellia oil, andginger oil of the drug composition can comprise one or more of thesematerials as disclosed herein. For example, the lecithin can besunflower lecithin, the boswellic acid component can comprise AKBA, theboswellia oil can comprise boswellia serrata oil, and the ginger oil cancomprise ginger root oil.

In some aspects, the invention provides methods for treatinginflammation. In some aspects, the invention provides methods fortreating an inflammatory disorder. The methods can be practice byadministering to a subject an effective amount of the drug compositionto a subject in need of treatment for inflammation or an inflammatorydisorder. As used herein, the term “subject” refers to a mammal,including but not limited to humans, non-human primates, cattle, sheep,dogs, cats, rats, mice, horses, goats, pigs or poultry (e.g. chickens,ducks, and geese). The inflammatory disorder can be an inflammatorydisorder that is mediated by macrophage activity, wherein administrationof the drug composition inhibits macrophage pro-inflammatory activity.The inflammatory disorder can be sepsis-related multiple organdysfunction/multiple organ failure, microbial infection, acutebrain/lung/hepatic/renal injuries, neurodegenerative disorders,tumorigenesis, osteoporosis/osteonecrosis, cardiovascular disease (e.g.atherosclerosis), metabolic diseases, Type II diabetes, localizedinflammation, and autoimmune diseases. The inflammatory disorder can bea gastrointestinal disorder. The gastrointestinal disorder can begastroparesis, post-operative ileus or inflammatory bowel disease. Theautoimmune disease can be rheumatoid arthritis.

In some aspects, the invention provides a method for treating melanomain a subject in need thereof comprising administering to the subject adrug composition as disclosed herein. The term “melanoma” includesprimary melanoma and metastatic melanoma. In certain embodiments, asubject is successfully “treated” for melanoma according to the methodsof the present invention if the patient shows one or more of thefollowing: a reduction in the number of, or complete absence of, canceror tumor cells; a reduction in the tumor size; inhibition of, or anabsence of, cancer or tumor cell infiltration into peripheral organsincluding, for example, the spread of tumor into soft tissue and bone;inhibition of, or an absence of, tumor metastasis; inhibition of, or anabsence of, tumor growth; relief of one or more symptoms associated withmelanoma; reduced morbidity and mortality; improvement in quality oflife; reduction in tumorigenicity, tumorigenic frequency, or tumorigeniccapacity of a tumor; reduction in the number or frequency of cancer stemcells in a tumor; reduction in the number or frequency of tumorinitiating cells in a tumor; differentiation of tumorigenic cells to anon-tumorigenic state; or some combination of effects.

In methods the of treatment disclosed herein, the drug composition canbe administered to the subject topically, orally, sublingually,buccally, intra-ocularly, intravenously, intramuscularly,intra-arterially, by suppository, intranasally, subcutaneously,parenterally, intravaginally, rectally, by inhalation, or a combinationthereof. In a preferred embodiment, the drug composition is administeredorally.

In some aspects of the invention, one or more of the active agents orbioenhancers of the drug composition are organic. As used herein, theterm “organic” refers to, relating to, yielding, or involving the use offood produced with the use of feed or fertilizer of plant or animalorigin without employment of chemically formulated fertilizers, growthstimulants, antibiotics, or pesticides. In some aspects, organiccomponents for use with the drug composition are certified by anorganization that is approved and recognized by the USDA NationalOrganics Program.

The drug compositions can comprise one or more agents for improving thepalatability of the composition. For example, the drug compositions cancomprise sweeteners, aromatic compounds, flavourings, or a combinationthereof. Similarly, the drug compositions can comprise agents toincrease the antioxidant and/or nutritional value of the compositions.Such agents include, but are not limited to, vitamins, minerals,proteins, amino acids, and carbohydrates.

In some aspects of the invention, the drug composition can beencapsulated. Such encapsulation may be accomplished via liposomes,nanospheres and/or micelles. Various methods of preparing theencapsulation of the drug compositions are described, for example, inU.S. Pat. Nos. 3,932,657, 4,235,871, 4,311,712, and 5,013,556, thedisclosures of which are incorporated herein by reference for allpurposes. In one non-limiting embodiment, the liposome comprises alecithin liposome.

The present disclosure is further described in the light of thefollowing non-limiting examples which are set forth for illustrationpurpose only and not to be construed for limiting the scope of thedisclosure.

Example 1—Drug Formulation

Turmeric extract was standardized to 95% curcuminoids, boswellia extractwas standardized to 30% AKBA, and ginger extract was standardized to 20%gingerols. These components were combined to achieve a compositioncomprising 50% of the turmeric extract, 30% of the boswellia extract,and 20% of the ginger extract. The mixture was dried to a powder. Thisdrug composition was used in the following example.

Example 2—Drug Effects on Inflammation and Melanoma Cell Viability CellCulture

The murine macrophage RAW 264.7 and B16-F10 melanoma cell line waspurchased from the National Centre for Cell Science (Pune, India). RAW264.7 cells were cultured in Dulbecco's modified Eagle's medium (DMEM;GIBCO Inc., NY, USA) supplemented with 100 U/ml of penicillin, 100 lg/mlof streptomycin and 10% fetal bovine serum (FBS; GIBCO Inc., NY, USA).The cells were incubated in an atmosphere of 5% CO2 at 37° C. and weresub-cultured every 3 days.

MTT Assay for Assessment of Cytotoxicity

The effect of the drug composition on RAW 264.7 and B16-F10 melanomacell line was observed by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. Cells were counted on haemocytometer and5000 cells/well were plated in 96 well plate in 100p of complete media(containing 10% of FBS). Cells were treated with differentconcentrations (0.1 0.2 0.4 0.6 0.8 1.0 and 1.2 mg/mL) of the drugcomposition for 48 hr. After the stipulated time, MTT solution (5 mg/ml)was added to each well and incubated for 3 hr. The purple precipitate offormazan was dissolved in 150 μl of DMSO (Sigma-Aldrich) by propermixing. The colour absorbance of each well was recorded at 570 nm inMultiscan EX reader with a reference serving as blank. Then, IC50 valueof the drug composition was calculated.

Morphological Study Under Phase Contrast Microscopy

B16-F10 melanoma cells were treated with different concentrations (0.4and 0.8 mg/mL) of the drug composition for 48 hr. Following incubation,the cells were observed under phase contrast microscope at 20×magnification.

Determination of NO Production

After pre-incubation of RAW 264.7 cells (1.5×10⁵ cells/ml) with LPS (1μg/ml) plus samples at 37° C. for 24 h, the quantity of nitriteaccumulated in the culture medium was measured as an indicator of NOproduction (Lee et al., 2007). Briefly, a 100 μl of cell culture mediumwas mixed with 100 μl of Griess reagent (1% sulphanilamide and 0.1%naphthylethylenediamine dihydrochloride in 2.5% phosphoric acid), themixture was incubated at room temperature for 10 min, and the absorbanceat 540 nm was measured in a microplate reader (Mutiscan EX). Freshculture medium was used as a blank in every experiment.

Results Cytotoxicity and Inhibitory Effect of NO Production of the DrugComposition

The cytotoxic effect of the drug composition was assessed by MTT assay.The drug composition did not influence the cytotoxicity, with only thehighest concentration (1.2 mg/mL) affecting cell viability at around24.3% of RAW 264.7 cells (FIG. 1). In order to study the potentialanti-inflammatory effect of the drug composition, RAW 264.7 cells weretreated with 1 μg/ml LPS with or without different concentration of thedrug composition. After 24 h, nitrite concentrations were determined asan indicator of NO production. As shown in FIG. 2, the drug compositionsignificantly inhibited the LPS-induced production of NO.

Reduction in Viability of B16-F10 Melanoma Cells

MTT assay was performed to evaluate the cytotoxic effect of the drugcomposition on B16-F10 melanoma cells were treated with differentconcentrations (0.1, 0.2, 0.4, 0.6, 0.8, 1.0 and 1.2 mg/mL) for 48 hr. Asignificant reduction of cell viability was seen in dose dependentmanner when compared with the control or vehicle treated B16-F10melanoma cells. The calculated IC50 value for 48 hr is 0.85 mg/mL (FIG.3).

Phase Contrast Microscopic Observations of B16-F10 Melanoma Cells afterTreatment

Morphological alterations were observed in treated B16-F10 cells incomparison to the control or vehicle treated cells. In case of controlcells, the shape of B16-F10 cell is polygonal but after treatment withdifferent concentrations of the drug, the shape became spherical andcell numbers decreased (FIG. 4).

Statistical Analysis

All the measurements were made in triplicate and all values wererepresented as means standard error. Statistical analysis was performedby one-way ANOVA followed by Student ‘t’ test using GraphPad softwareprism 7.01.

DISCUSSION AND CONCLUSION

Lee et al., 2011; Poltorak et al., 1998 and Kanno et al., 2006. Reportedthat macrophages play critical roles in immune reaction, allergy, andinflammation. These cells induce inflammatory reaction, and initiate andmaintain specific immune responses by releasing different types ofcytokines. Macrophage activation by lipopolysaccharides (LPS), which arederived from gram-negative bacteria cell walls, results in the releaseof several inflammatory mediators including nitric oxide (NO),cyclooxygenase (COX)-2, interleukin (IL)-6, IL-1b, and tumor necrosisfactor (TNF)-a. Over-expression of the inflammatory mediators inmacrophage is involved in many inflammation related diseases, such asatherosclerosis, rheumatoid arthritis, autoimmune diseases. Thus,inhibition of inflammatory mediators produced by macrophages is crucialfor managing inflammatory diseases.

In conclusion, from our preliminary data we found that the drugcomposition showed significant inhibition of NO in LPS stimulated RAW264.7 cells. In addition, it also showed anticancer effect on B16-F10melanoma cells.

1. A composition comprising about 50% turmeric extract, about 30%boswellia extract, and about 20% ginger extract.
 2. The composition ofclaim 1, wherein the turmeric extract is standardized to about 95%curcuminoids.
 3. The composition of claim 2, wherein the turmericextract comprises about 1% lecithin and about 1% turmeric oil.
 4. Thecomposition of claim 1, wherein the boswellia extract is standardized toabout 30% boswellic acid.
 5. The composition of claim 4, wherein theboswellic acid comprises AKBA.
 6. The composition of claim 4, whereinthe boswellia extract comprises about 1% lecithin and about 1% boswelliaoil.
 7. The composition of claim 1, wherein the ginger extract isstandardized to about 20% gingerols.
 8. The composition of claim 7,wherein the ginger extract comprises about 1% lecithin and about 1%ginger oil.
 9. The composition of claim 1, wherein the compositioninhibits nitric oxide production by about 10% at a concentration of 0.1mg/ml.
 10. The composition of claim 1, wherein the drug composition isencapsulated within a liposome.
 11. The composition of claim 1, whereinthe drug composition is formulated as a powder, pill, tablet, pellet,capsule, thin film, solution, spray, syrup, linctus, lozenge, pastille,chewing gum, paste, vaporizer, suspension, solution, emulsion, ointment,cream, lotion, liniment, gel, drop, topical patch, buccal patch, bead,gummy, gel, sol, or injection.
 12. A method for treating inflammation,the method comprising administering to a subject in need thereof acomposition comprising about 50% turmeric extract, about 30% boswelliaextract, and about 20% ginger extract.
 13. The method of claim 12,wherein the turmeric extract is standardized to about 95% curcuminoids.14. The method of claim 13, wherein the turmeric extract comprises about1% lecithin and about 1% turmeric oil.
 15. The method of claim 12,wherein the boswellia extract is standardized to about 30% boswellicacid.
 16. The method of claim 15, wherein the boswellic acid comprisesAKBA.
 17. The method of claim 15, wherein the boswellia extractcomprises about 1% lecithin and about 1% boswellia oil.
 18. The methodof claim 12, wherein the ginger extract is standardized to about 20%gingerols.
 19. The method of claim 18, wherein the ginger extractcomprises about 1% lecithin and about 1% ginger oil.
 20. The method ofclaim 12, wherein the composition is encapsulated within a liposome.